1. Field of the Invention
In general, analysis apparatus, such as spectroscopic analysis apparatus are used to investigate the composition of an object to be examined. In particular, analysis apparatus employ an analysis, such as a spectroscopic decomposition, based on interaction of the matter of the object with incident electromagnetic radiation, such as visible light, infrared or ultraviolet radiation.
2. Description of the Related Art
Conventional analysis apparatus is known from the U.S. Pat. No. 6,069,690, incorporated herein by reference The known analysis apparatus concerns a dual mode integrated laser imaging and spectral analysis system, which is used to view and analyse defects on a work piece such as a semiconductor wafer. This known analysis apparatus has two operating modes, namely a scanned imaging mode and a stop scan spectral analysis mode. During the scanned imaging mode a monitoring beam in the form of a laser beam is emitted and the target region is imaged. Separately from the imaging, in the stop scan mode, the laser beam is employed for excitation and spectral analysis can be carried out.
An object of the invention is to provide an analysis apparatus which supplies an analysis of a target comprised in the object to be examined more reliably than the known analysis apparatus.
This object is achieved by an analysis apparatus according to the invention wherein the excitation period and the monitoring period substantially overlap.
During the overlap of the excitation period and the monitoring period the excitation of the target region and the monitoring of the target region occur simultaneously and/or alternatingly. Because the target region is imaged together with the excitation, an image is formed displaying both the target region and the excitation area. On the basis of this image the excitation beam can be very accurately aimed at the target region. Consequently, the excitation beam generates scattered radiation almost exclusively in the target region, or at least the target region is included in the area that is excited by the excitation beam. The scattered radiation from the target region is detected and the composition of the target region is derived from the scattered radiation.
In particular the analysis apparatus performs spectral analysis of the scattered radiation from the target region to determine the material composition. For the excitation beam various types of electromagnetic radiation, notably ultraviolet radiation, visible light or infrared radiation can be employed. Owing to the excitation scattered radiation may be generated by different physical mechanisms of interaction of the excitation beam with matter in the excitation area. Scattered radiation includes elastically scattered radiation, inelastically scattered radiation or other types of emission such as fluorescence or phosphorescence, or scattering by multi-photon excitation or non-linear scattering generated by the excitation beam. For example Raman scattering or fluorescence due to single or multi-photon excitation may be generated by the excitation.
In particular, the spectroscopic analysis apparatus according to the invention is advantageously employed to examine in vivo the composition of blood in a capillary vessel of a patient to be examined. In this application the target area is the capillary vessel which is typically located about 50-150 xcexcm under the skin surface. This capillary vessel is imaged by the monitoring system and the excitation beam is accurately directed to the capillary vessel. For example near-infrared radiation is used for excitation of Raman scattering. The Raman scattered radiation is spectroscopically analysed. It appears that the in vivo Raman spectra obtained with the spectroscopic analysis apparatus of the invention have about the same quality, spectral resolution and signal-to-background ratio, as for Raman spectra of human blood obtained in vitro.
Monitoring of the target region can be performed in several ways, preferably the monitoring beam is employed to illuminate the target region with its surroundings and image the target region with its surroundings by way of the reflected monitoring beam from the target region. The reflected monitoring beam may return from the target region and its surroundings either by specular or diffuse reflection and by back-scattering. As an alternative, in the event the object to be examined is to some extent transparent for the monitoring beam, the monitoring beam having been transmitted through the target region may be employed to image the target region. The excitation beam having passed through the target region may be imaged onto the image of the target region in order to display the excited area in the image of the target region.
Preferably, the analysis apparatus according to the invention includes a beam combination unit, which directs the monitoring beam and the excitation beam to the target region. The beam combination further separates the reflected monitoring beam and at least part of the reflected excitation beam from the scattered radiation. Hence, the scattered radiation on the one hand and the reflected monitoring beam with at least part of the reflected excitation beam can be detected separately. From the detected scattered radiation there is information obtained relating to the local composition at the target region. For example the local composition concerns the molecular composition in a small area. The reflected monitoring beam is used to image the target region. The reflected excitation beam can also be imaged in the image of the target region. The image of the target region then shows the actual target and also the location where the excitation is done relative to the target. On the basis of the image it is easy to direct the excitation beam exactly onto the target.
In an example of an analysis apparatus of the invention, the beam combination unit reflects the scattered radiation preferably substantially to a detection system. Several examples of detection systems may be employed depending on the type of scattered radiation. A Raman-spectrometer may be employed for detecting inelastically scattered radiation, notably such as Raman scattered radiation. To detect fluorescently scattered radiation a fluorescence spectrometer is used. The beam combination unit transmits the reflected monitoring beam preferably substantially and transmits the reflected excitation beam from the target region at least partially to an imaging system incorporated in the monitoring system to form the image displaying the target region and the excitation area. In an other example of the analysis apparatus of the invention the beam combination unit reflects the reflected excitation beam from the target region at least partially and the reflected monitoring beam preferably substantially to form the image displaying the target region and the excitation area. In that example the scattered radiation due to the excitation beam is preferably substantially transmitted to the detection system. In the examples of the analysis apparatus of the invention as defined herein the scattered radiation can be separated spatially from the reflected monitoring beam and the reflected excitation beam.
In a further example of the analysis apparatus of the invention, the scattered radiation is separated in time from the reflected monitoring beam and the reflected excitation beam. The same spatial aperture is shared by these beams alternatingly in that in separate time slots, the scattered radiation is passed to the detector system and the reflected monitoring beam is passed to the imaging system, respectively. In a preferred embodiment of the analysis apparatus of the invention alternatingly sharing the spatial aperture through which the various beams, the scattered radiation, the reflected monitoring beam and the reflected excitation beam, is achieved in that the partial transmission alternates partial reflection of these respective beams. In a simple way the alternation of reflection and transmission is achieved by moving a reflector/transmission unit having reflective and transmissive sections that are moved relatively to the various beams. Preferably a rotatable reflector/transmission unit is used which turns the reflective and transmissive sections into and out of the various beams.
Another preferred embodiment of the analysis apparatus of the invention employs yet another variation of sharing the spatial aperture. In this embodiment the reflector/transmission unit of the beam combination unit has a reflective section, preferably a small portion in the centre of the reflector/transmission unit that directs the excitation beam to the excitation area. The reflective section is substantially opaque for the reflected monitoring beam The reflector/transmission unit has a transmissive section, preferably a larger area surrounding the reflective section, which transmits the reflected monitoring beam to the imaging system. The excitation beam is focussed onto the reflective section and reflected and preferably converged into a parallel excitation beam and focussed onto the target region. Part of the (reflected) monitoring beam is intercepted by the reflective section as it is opaque for the (reflected) monitoring beam and the reflective section causes a low-brightness spot in the image formed by the imaging system. For example, the monitoring beam is scanned over the reflector/transmission unit so that also the reflected monitoring beam is scanned over the reflector transmission unit. The image formed by the imaging system will then comprise a low-intensity spot corresponding to the opaque reflective section, which indicates where the excitation beam is directed. The image also shows the target region so that is easy to bring the target area into correspondence with the excited area.
In a variation of the analysis apparatus of the invention the excitation beam and the scattered radiation are substantially transmitted through the transmissive section. This transmissive section is for example formed as a small opening in the reflector/transmission unit, preferably in the centre of the reflector/transmission unit. The reflective section is substantially reflective for the monitoring beam and the reflected monitoring beam. For example the reflective section is formed as the surrounding area around the small opening that acts as the transmissive section. This opening is imaged as a low-brightness spot in the image and marks the excitation area that is reached by the excitation beam.
These reflection and transmission properties are achieved by means of wavelength dependent filters and reflectors. These reflectors and filters are discussed in more detail with reference to the detailed embodiments and with reference to the drawings. Notably, the separation of respective beams is achieved because the scattered radiation, such as Raman scattered radiation or multi-photon fluorescence, has a wavelength that is different from the wavelengths of excitation beam and of the monitoring beam
In a further preferred embodiment the monitoring beam and the excitation beam are derived from a single radiation source. Preferably an infrared or optical laser is used to generate the output beam which is then split by a beam splitter, such as a dividing prism or a semi-transparent mirror into the monitoring beam and the excitation beam. This preferred embodiment has a relatively simple and less expensive set-up which only uses a single radiation source.
Preferably, the analysis apparatus comprises a monitoring system including a confocal optical imaging system, such as a confocal video microscope. Good results are particularly obtained with a confocal laser scanning video microscope. The confocal optics focuses the monitoring beam onto a focal plane at the target region and also images this focal plane on the imaging system, notably on to an image pick-up device. The confocal optics of the monitoring system achieves that mainly, or even almost exclusively a region of the object to be examined is imaged where the monitoring beam is focussed. Thus, by changing the focussing of the monitoring beam, the region being imaged can be selected and the image is not or hardly at all disturbed by adjacent portions of the object to be examined. According to the invention also the detection system that receives the scattered radiation is confocally related to the confocal video microscope. The confocal detection accomplishes that the scattered radiation that reaches the detector mainly, or essentially only, originates from the focus of the excitation beam. Preferably, a detection pin-hole is arranged in front of the detector and the scattered radiation is focussed on this detection pin-hole while the excitation beam is focussed on the target region. In particular a fibre entrance can function as the detection pin-hole. Hence, this preferred embodiment of the analysis apparatus according to the invention focuses the monitoring beam and the excitation beam onto the target area and forms an image of the focal plane of the monitoring beam and detects scattered radiation essentially only from the focal plane of the excitation beam. Since the focal planes of the excitation beam and the monitoring beam coincide, scattered radiation is received at the detection system from the target area being monitored in the image formed by the reflected monitoring beam.
Further, the confocal video microscope can adjust the position of the focal plane along the direction normal to the focal plane. Thus, the depth of the target region can be adjusted. During displacement of the focal plane into the depth of the object the confocal relation of the detection system with the confocal optical imaging system achieves that the scattered radiation received by the detector originates from the target region. This preferred embodiment is in particular advantageous to analyse blood in a capillary vessel under the skin surface of a human or animal to be examined.
In a further preferred embodiment of the analysis apparatus according to the invention, the orthogonal polarised spectral imaging arrangement is employed in the monitoring system. In this embodiment a spectrally relatively narrow polarised monitoring beam is employed. The reflected monitoring beam is imaged through an analyser at orthogonal polarisation direction relative to the polarisation direction of the monitoring beam. Hence, substantially only multiply diffused depolarised radiation reaches the imaging system to form a substantially uniform background. Portions in the object to be examined, notably in the target region that substantially absorbs the spectrally narrow monitoring beam are then imaged as low brightness in the image. The monitoring system also images the reflected excitation beam in the same image so that the target region and the excitation area are easily brought into correspondence. Orthogonal polarisation spectral imaging is known per se from the paper xe2x80x98Orthogonal polarisation spectral imaging: a new method for study of microcirculationxe2x80x99 by W. Groner et al. in Nature Medicine 5(1999)1209-1213 to study the morphology of the vessel structure.
Other suitable options for the monitoring systems are for example an optical coherence tomography (OCT) arrangement, an optical Doppler tomography (ODT) arrangement, a photo-acoustic imaging (PAI) arrangement, or a multiphoton microscopy (MPM) arrangement. Notably, the OCT, ODT and PAI arrangements give good results for monitoring blood vessels or other target areas that lie deeper, up to several millimeters, under the skin surface. The MPM arrangement in conjunction with confocal imaging provides a high resolution where details of 3-5 xcexcm are rendered well visible. The MPM arrangement is further suitable for imaging details at a depth up to 0.25 mm.
In a further preferred embodiment an acousto-optic modulator is included in the beam combination unit. In the acousto-optic modulator an acoustic wave is generated which causes diffraction of the various beams. Both a standing as well as a running acoustic wave may be employed. A running acoustic wave is employed in combination with a scanning monitoring beam. In particular the zeroth order diffracted excitation beam and the first order diffracted monitoring beam reach the target region. The first order reflected monitoring beam and the first order diffracted scattered radiation and the first order reflected excitation beams are passed to the imaging system. The zeroth order diffracted scattered radiation is passed to the detection system.
In a further preferred embodiment of the analysis apparatus of the invention the excitation beam is scanned transversely to the longitudinal axis of the target region. This preferred embodiment is especially advantageous for examining elongate target regions of which the longest size is along the longitudinal axis. As the excitation beam scans across the elongate target area, the probability of receiving at least in part scattered radiation from the target region is greatly enhanced.
A further object of the invention is to provide a method of spectral non-invasive analysis of the composition of blood in vivo. It is noted that in spite of numerous attempts to achieve this object made over the last decades, these attempts were unsuccessful in that satisfactory signal-to-background ratio of the spectrum of blood in vivo could not be obtained. Notably as to non-invasive glucose monitoring L. Heinemann et al, in xe2x80x98Diabetes Technology and Therapeutics, Vol. 2, pp 211-220, 2000) note that xe2x80x9cDespite the more than 20 years of intensive research, numerous publications and encouraging announcements, until now no reliable system has been developedxe2x80x9d In the paper xe2x80x98Capillary blood cell velocity in human skin capillaries located perpendicularly to the skin surface: measured by a new laser Doppler anemometer.xe2x80x99 by M. Stxc3xccker et al. in Microvascular Research 52(1006)188 only success is reported as to measurement of the velocity of blood cells, but not on the composition of the blood. From the U.S. Pat. No. 5,615,673 it is known per se to employ Raman spectroscopy to blood and tissue. However, this known method detects Raman scattered radiation from both capillary vessels at issue as well as to a large extent from tissue between the skin surface and the capillary vessel and hence the detected Raman spectra do not hava a satisfactory signal-to-background ratio of the spectrum of blood in vivo.(In the international application WO92/15008 a method for investigating tissue by means of Raman spectroscopy is disclosed. The U.S. Pat. No. 5,372,135 mentions analysis of blood from differential optical absorption spectra. In the paper xe2x80x98A Noninvasive Glucose Monitor: Preliminary Results in Rabbitsxe2x80x99 by Mark S. Borchert, et al in Diabetes Technology and Therapeutics Volume 1, Number 2, 1999, application of Raman spectroscopy to a rabbit""s eye is discussed. The U.S. Pat. No. 5,553,616 discloses an analysis by means of an artificial neural network discriminator of Raman scattering intensity to determine glucose concentration in e.g. the skin of an index finger. In that known analysis satisfactory signal-to-background ratio of the spectrum of blood in vivo could not be obtained. Notably this appears to be due to lack of accurate detection of the baseline. Moreover, faithful representation of glucose blood level from measurements at the eye appears to be questionable. Hence, these known methods of analysis do not provide a sufficiently high signal-to-noise ratio. Notably in the overview paper xe2x80x98Overview of non-invasive glucose measurement using optical techniques to maintain glucose control in diabetes mellitusxe2x80x99 by R. W. Waynant et al. in the Leos Newsletter Volume 12 Number 2 April 1998 it is remarked that: xe2x80x98Current instrumentation lacks specificity due to substantial chemical and physical interferences.xe2x80x99
The present invention satisfies the long-felt need for accurate in vivo analysis of human or animal blood, an is particularly suitable to obtain accurate measurement of the glucose content in human blood in viva. Preferably, the excitation beam is an infrared laser beam and the scattered radiation is Raman scattered infrared radiation.